All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
The degeneration and/or death of cells in the nervous system are a major factor in many diseases and medical conditions. Such diseases and conditions include traumatic brain injuries, traumatic spinal cord injuries, stroke, hypoxia or ischemia related to decreased neural perfusion, secondary to cardiac arterial bypass graft surgery (CABG), neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases as well as decrease in neuronal function associated with normal aging processes. It is of interest to prevent or decrease such cell death and degeneration and to minimize the loss of neural function.
Nutritional status is an important determinant of risk for neurodegenerative disease and cognitive decline among old adults. Deficiencies of nutrients including omega-3 fatty acids, B-vitamins, and antioxidants can exacerbate or increase susceptibility to pathological processes such as glutamate-induced neuronal injury that is a prominent feature of Alzheimer's and Parkinson's disease, epilepsy and stroke. Theoretically, nutrients and natural compounds found in edible fruits can mitigate some of this damage and can therefore play an important role in a comprehensive approach to neuro-protection. However, actionable evidence for the neuroprotective activity of specific edible fruits is limited. It was thus the goal of the inventors to develop edible fruit or extracts as nutraceutical products to protect the aging brain.
The genus Passiflora consist of about 500 species. Fifty to sixty species bear edible fruits, however, only a few are of commercial value, mainly P. edulis. The round to oval fruit contains several hundred seeds, each surrounded by an aromatic juicy aril. This semi-woody perennial climbing vine produces large attractive flowers resembling a crucifix to some Spanish monks who named it “passion fruit”. Two major varieties, purple (P. edulis Sims f. edulis) and yellow (P. edulis Sims f. flavicarpa Deg.) passion fruit, are grown worldwide, mainly for juice production and fresh fruit. In Israel, the commercially grown cultivar “Passion Dream” (PD) is an F1 hybrid of a cross between these two varieties showing a combination of agriculturally important traits.
P. edulis has an extremely short juvenile phase in comparison to other fruit-providing species. The PD cultivar is self-fertile and from its seeds one can screen the F2 population from the original cross of the two varieties (yellow and purple). In the last 5 years the inventors have characterized close to 1000 F2 seedlings and in this F2 population there is variation in many important aspects including flowering time, climacteric behavior, taste and fruit size. While many traits are likely controlled by more than one gene, the color of fruit might be controlled by one locus since about 75% of the seedlings have purple colored fruit and 25% are yellow-green.
The unique aroma and flavor of passion fruit encouraged intensive research leading to the characterization of a broad spectrum of volatile constituents and attempts to synthesize them commercially [Murray, K., et al. (1972) Aust. J. of Chem. 25(1921-1933); Prestwich, G. D., et al. (1976) Tetrahedron 32:2945-2948; Scafato, P., et al. (2009) Chirality 21:176-182; Tominaga, T. and Dubourdieu, D. (2000) J. Agric. Food. Chem. 48:2874-2876; Werkhoff, P., et al. (1998) J. Agric. Food Chem. 46:1076-1093]. There is currently a revival of interest in the pharmaceutical industry, especially in Europe, in the use of the glycoside passiflorine, especially from P. incarnata L., as a sedative or tranquilizer. Italian chemists have extracted passiflorine from the air-dried leaves of P. edulis, and in Madeira, the juice of passion fruits is given as a digestive stimulant and treatment for gastric cancer.
The inventors have looked at variation among the select new lines they characterized with regard to the content of volatiles, using GCMS. Interesting differences were identified in levels of a group of thiol esters (sulfur containing volatiles) which were previously identified in passion fruit and shown to contribute to the distinct aroma associated with it: 3-mercaptohexyl acetate (3MHA), 3-mercaptohexyl butanoate and 3-mercaptohexyl hexanoate [Werkhoff, P., (1998) ibid.; Engel, K. and Tressl, R. (1991) J. Agric. Food Chem. 39:2249-2252]. Attempts were made by others to improve wine aroma by increasing levels of 3MHA [Swiegers, J., et al. (2007) Yeast 24:561-574]. A strong influence of the configuration of these chiral volatiles on their sensory properties has been demonstrated [Werkhoff, P., (1998) ibid.].
The inventors have screened such a population of passion fruits for lines that provide agriculturally important benefits (such as taste, yield, season), and examined the neuroprotective activity of these lines as well as the neuroprotective effect of the parent PD line. The surprising neuroprotective benefits of the passion fruit extracts, and specifically of the novel 428 cultivar, the inventors disclose here are applicable to many neurologic disorders and traumas.
More specifically, it is well established that elevated levels of extracellular glutamate are associated with neuronal damage and degeneration in brain disorders, including epilepsy, stroke and Parkinson's disease [Coyle, J. T. and Puttfarcken, P. (1993) Science 262(5134):689-695]. There are two defined mechanisms of glutamate cytotoxicity: the excitotoxicity and excitotoxicity-independent pathways. In the excitotoxicity pathway, superactivation of glutamate ionotropic receptors is followed by an increase in free intracellular calcium levels, and the generation of reactive oxygen species (ROS) in the cell [Savolainen, K. M., et al. (1998) Toxicol. Lett 102-103:363-367]. The excitotoxicity-independent process relies on an imbalance between oxidants and antioxidants in the cell (oxidative stress). Inhibition of cystine uptake via the xe− cystine/glutamate antiporter system by glutamate leads to depletion in glutathione (GSH), the major intracellular antioxidant, and excessive production of ROS [Murphy, T. H., et al. (1989) Neuron 2(6):1547-1558]. There are evidence that these two pathways are activated in neuronal injuries [Tan, S., et al. (2001) Curr. Top. Med. Chem. 1(6):497-506]. An established in-vitro model system for investigating glutamate-oxidative-stress-mediated cell death is the immortalized mouse hippocampal cell line HT4, which phenotypically resembles neuronal precursor cells. Depletion in GSH and increased production of ROS in these cells are preceded by activation of c-Src and 12-Lox [Sen, C. K., et al. (2000) J. Biol. Chem. 275(17):13049-13055].
The inventors treated HT4 neuronal cells with chemicals that normally cause cell death by generating low levels of hydrogen peroxide flux or elevated levels of extracellular glutamate. Whereas extracts from the parent line gave relatively high protection, the F2 population segregated for protective ability with some lines giving low protection while other lines (such as line 428) giving even higher protection than both the PD parent line and NAC (N-acetyl cysteine), a known neuroprotective antioxidant. Thus, the inventors can breed and select lines presenting exceptionally high levels of neuro-protection. It should be noted that the inventors demonstrate here for the first time improved neuroprotective properties of passion fruit, specifically of the novel cultivar 428.
Age-associated neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and vascular dementia are leading causes of mortality and disability worldwide. They do not have a cure and pose an enormous burden to caregivers, the health care system and society at large. With the aging of the world's population, and in the absence of effective pharmacological interventions, this problem is only expected to grow. Thus, there is an urgent need for new safe and efficacious intervention to prevent or mitigate neurodegenerative disease. The inventors provide herein passion fruit extracts, and specifically 428 and PD extracts, as neuroprotective functional foods, nutraceuticals, and for production of derivative pharmacological agents.
Thus, one object of the invention is providing a method for protection of neural cells from damage or deterioration in neural cell function using extracts of passion fruits. The invention further provides methods for prophylaxis, treatment, amelioration, inhibition or delaying the onset of neuro-pathological conditions using extracts of passion fruits.
Another object of the invention is providing neuroprotective compositions comprising passion fruit extracts, specifically of the new (428) and the parent PD cultivars extracts.
Yet another object of the invention is providing neuroprotective nutraceutical compositions comprising passion fruit extracts, specifically the newly-established 428 and PD cultivars extracts.
A further object of the invention is providing neuroprotective passion fruit extracts, specifically new (428) and PD cultivars extracts and uses thereof in treating neuro-pathologies.
These and other objects of the invention will become apparent as the description proceeds.